建立发酵液中冠毒素检测的薄层色谱法

Establishing the methods of identify and determination of coronatine from fermentation broth by TLC

目的:研究菌株发酵样品中冠毒素的薄层色谱分离条件,建立定性、粗定量测定发酵液中冠毒素的方法。方法:薄层色谱法,以微量圆环法等逐步找到最佳溶剂系统,并选用适当的参照物进行对比分析。以化学显色和相对比移值来进行定性分析,再以斑点面积测量法进行定量分析。结果:建立了以乙醇-乙酸-丙酮-氯仿(体积比为0.3:0.1:3:15)的混合溶剂为最佳溶剂系统,硅胶 G 溥层板为层析板,香兰素-浓硫酸溶液为显色剂,3,5-二羟基甲苯(DHT)溶液为参照物,以冠毒素特有的桔黄色斑点及其与参照物的相对比移值——R_(st)值(约0.55)进行定性分析,运用冠毒素点样量(Y)和冠毒素与参照物斑点的面积比值(X)之间的标准曲线 Y=3.28X-0.30,Y 线性范围0.25～2.0μg,粗定量测定发酵液中冠毒素的含量。此方法操作简单、方便,在产冠毒素菌株的筛选和发酵样品的分析中得到很好的应用。

Objective：The paper has carried out the condition of coronatine apart and established the methods of determining coronatine qualitatively and semi - quantitatively from fermentation brothe by TLC. Methods： The TLC, using micro - cirque method for solvent system, chemi - develop method and relative Rf value for qualitative analy-sis, spot area measurement for semi - quantitative analysis. Results： The TLC ways has estabilished, which were eth-anol -acetic acid -acetone -chloroform （0.3：0. 1：3：15 in volume ratio）as a solvent system, silica gel G plate as a chromatography plate, and vanillin - sulfuric acid as a developing solvent,3,5 - dihydroxytoluene （DHT） as a marker,both of distinctive saffron yellow spot of corontine and relative Rf value（Rst 0.55 approximately）of the marker as qualitative analysis, standard curve Y = 3.28X - 0.30 （ X-spot area ratio of coronatine and the marker, Y-corona- tine quantity of sample application, the linear concentration range of Y is 0.25 - 2.0 μg. ） as semi - quantitative to mensurating coronatine from formentation broth. The TLC is simple and convenient. There was common practical application of the TLC methods for screening coronatine strains and formentation sample analysis.